The specific aims of this project are: (1) to obtain an in vitro system capable of synthesizing phytosphingosine, and (2) to determine the metabolic fate of radioactively-labelled phytosphingosine in cultures of Hansenula ciferrii. There are only a limited number of drugs effective in the treatment of human yeast infections. Of those available, only a few are specific for yeast, several produce adverse side effects in humans, and most are limited to treatment of topical infections. Since phytosphingosine appears to be central, and highly specific, to plant and fungal membrane metabolism, a more complete description of its biosynthesis will provide a basis for selection or synthesis of inhibitors of the hydroxylation reaction that is unique to the formation of phytosphingosine. The availability of these inhibitors will aid in determining the biochemical function of phytosphingosine and, ultimately, in developing therapeutic agents for the treatment of pathogenic yeast infections. In addition, a more thorough understanding of phytosphingosine's biochemical role in yeast will provide a better understanding of the role of sphingolipids in less accessible systems, such as the human nervous system. Phytosphingosine and its derivatives appear to play an important, but as yet poorly defined, role in membrane structure and function in fungi and plants. The yeast Hansenula ciferrii synthesizes large amounts of phytosphingosine, thereby providing an attractive experimental system in which to study the synthesis and metabolism of this lipid. Since all yeasts that have been examined synthesize phytosphingosine, knowledge gained from studies in Hansenula will be of general applicability. Incubations containing cell-free homogenates of Hansenula ciferrii and various potential substrates, cofactors, and stabilizing agents will be carried out; phytosphingosine formation will be measured by gas-lipid chromatography. Radioactively-labelled phytosphingosine will be prepared and purified. This labelled lipid will be incubated with Hansenula cultures; the radioactively- labelled sphingolipid base and/or its metabolites will be isolated from yeast cells and spores and identified.